Various publications or patents are referred to in parentheses throughout this application to describe the state of the art to which the invention pertains. Each of these publications or patents is incorporated by reference herein.
One of the paramount goals of medical therapy is the efficient delivery of therapeutic substances to the site of disease. While some therapeutic substances can be delivered in free form, others require a carrier or vector in order to reach and enter their final destination, either due to their rapid clearance from the area of introduction or their inability to cross biological barriers, or due to their systemic toxicity. Delivery of substances to cells and tissues requires vectors which are efficient, flexible, easy to prepare and safe. Currently available methods for delivering substances to eukaryotic cells involve the use of either viral or non-viral vectors. Viral vectors are replication-defective viruses with part of their coding sequences replaced by that of a therapeutic gene. Although recombinant viruses are highly efficient gene delivery and expression vectors, they are currently limited to the delivery of nucleic acids and their safety profiles have not yet been established for medical use in humans.
Most non-viral delivery systems operate at the following levels: loading of the delivery vector with a substance of interest (e.g. proteins, peptides, nucleic acids, pharmaceutical or other therapeutic drugs), endocytosis, and in the case of gene delivery, nuclear targeting and entry. The major drawback of non-viral systems, such as liposomes, is their low delivery efficiency to cells (Chu et al., id.; Legendre and Szoka, 1992, Pharmaceut. Res. Vol.9, P.1235), presumably due to the absence of fusion-mediating molecules on the surface of the liposomes. A hybrid type of delivery system, the virosome, combines the efficient delivery mechanism of viruses with the versatility and safety of non-viral delivery systems. Virosomes are reconstituted envelopes without the infectious nucleocapsids and the genetic material that can be derived from a variety of viruses. These virosomes are functional, in that their membrane fusion activity closely mimics the well-defined low-pH-dependent membrane fusion activity of the intact virus, which is solely mediated by the viral fusion protein. Like viruses, virosomes are rapidly internalized by receptor-mediated endocytosis. In contrast to viral systems virosomes are safe, since the infectious nucleocapsid of the virus has been removed. Thus, virosomes represent a promising carrier system for the delivery of a wide variety of different substances, either encapsulated in their aqueous interior or co-reconstituted in their membranes. Co-reconstitution of different receptors within the virosomal membrane, furthermore, allows the targeting of virosomes to different cells or tissues. So far, virosomes are mainly used as vaccines by adding antigen onto the surface of the virosomes.
A major limitation of the protocol currently used to prepare virosomes is that it does not result in high encapsulation efficiency. At the lipid concentration at which virosomes are produced (˜1 mM lipid), and given their mean diameter of approximately 200 nm, less than 1% of the aqueous phase will be entrapped within the virosomes (Schoen et al., J. Liposome Res. 3: 767-792, 1993). Such low entrapment rates reduce virosome-mediated efficiency of drug or gene delivery to cells. The development of new/novel, more efficiently loaded vesicles that retain the advantageous fusion properties of virosomes, as well as methods of making, loading, and delivering them would thus be a highly desirable goal in the field of therapeutic drug, protein and gene delivery.